U.S. patent application number 10/536767 was filed with the patent office on 2006-02-16 for sample drying device as well as mass spectrometer and mass spectrometry system therewith.
Invention is credited to Minoru Asogawa, Masakazu Baba, Wataru Hattori, Noriyuki Iguchi, Hisao Kawaura, Toru Sano, Hiroko Someya, Kazuhiro Uda.
Application Number | 20060032071 10/536767 |
Document ID | / |
Family ID | 32463023 |
Filed Date | 2006-02-16 |
United States Patent
Application |
20060032071 |
Kind Code |
A1 |
Baba; Masakazu ; et
al. |
February 16, 2006 |
Sample drying device as well as mass spectrometer and mass
spectrometry system therewith
Abstract
A channel (103) is formed in a substrate (101) and a drying area
(107) comprising a plurality of pillars (105) is formed in one end
of the channel (103). A cover (109) is formed over the channel
(103), except the area above the drying area (107). When a sample
is introduced into the channel (103), it is guided to the drying
area (107) by capillary phenomenon. The drying area (107) is heated
by a heater (111) to evaporate the solvent for concentrating and
drying the solute.
Inventors: |
Baba; Masakazu; (Tokyo,
JP) ; Sano; Toru; (Tokyo, JP) ; Uda;
Kazuhiro; (Tokyo, JP) ; Kawaura; Hisao;
(Tokyo, JP) ; Iguchi; Noriyuki; (Tokyo, JP)
; Hattori; Wataru; (Tokyo, JP) ; Someya;
Hiroko; (Tokyo, JP) ; Asogawa; Minoru; (Tokyo,
JP) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W.
SUITE 800
WASHINGTON
DC
20037
US
|
Family ID: |
32463023 |
Appl. No.: |
10/536767 |
Filed: |
November 28, 2003 |
PCT Filed: |
November 28, 2003 |
PCT NO: |
PCT/JP03/15252 |
371 Date: |
May 27, 2005 |
Current U.S.
Class: |
34/60 ;
34/61 |
Current CPC
Class: |
H01J 49/04 20130101;
G01N 1/40 20130101 |
Class at
Publication: |
034/060 ;
034/061 |
International
Class: |
F26B 19/00 20060101
F26B019/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 29, 2002 |
JP |
2002-349246 |
Claims
1. A sample drying device comprising: a channel for a sample
flowing in said channel; and a sample drying area having an opening
communicating with said channel, wherein said sample drying area
comprises a fine channel narrower than said channel.
2. A sample drying device comprising: a main channel for a sample
flowing in said main channel; a plurality of side channels branched
from said main channel; and a sample drying area communicating with
said side channels, wherein said sample drying area has a fine
channel narrower than said side channels.
3. The sample drying device as claimed in claim 2, wherein said
sample contains multiple components and said main channel comprises
a separating portion to separate said components.
4. The sample drying device as claimed in any of claims 1 to 3,
wherein said sample drying area comprises a plurality of
protrusions separated each other.
5. The sample drying device as claimed in claim 4, wherein said
drying area has a shape so that the top of said sample drying area
projects from said opening.
6. The sample drying device as claimed in any of claims 1 to 3,
wherein said sample drying area is filled with multiple
particles.
7. The sample drying device as claimed in any of claims 1 to 3,
wherein said sample drying area is filled with a porous
material.
8. The sample drying device as claimed in any of claims 1 to 3,
wherein said sample drying area has a lid comprising a fine channel
communicating with said outside of said sample drying device.
9. The sample drying device as claimed in any of claims 1 to 3,
wherein said sample drying device comprises a temperature
controller for controlling a temperature of said sample drying
area.
10. A mass spectrometer comprising a sample drying area included in
said sample drying device as claimed in any of claims 1 to 3, as a
sample holder.
11. A mass spectrometry system comprising: separating unit
separating components in a biological sample by their molecular
sizes and properties; pretreatment unit pretreating said sample
separated by said separating unit including enzymatic digestion;
drying unit drying the pretreated sample components; and mass
spectrometry unit conducting mass spectrometry for the dried
sample, where in said drying unit comprises said sample drying
device as claimed in any of claims 1 to 3.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates to a sample drying device as well as
a mass spectrometer and a mass spectrometry system therewith.
[0003] 2. Description of the Related Art
[0004] Microchips capable of separating a protein or nucleic acid
have been intensely investigated and developed (Patent document 1).
On such a microchip, there is formed a feature such as a
micro-channel for separation by fine processing, whereby an
extremely small amount of sample can be introduced into the
microchip for separation.
[0005] However, in a separation process using a conventional
microchip, a component separated is obtained as a solution or
dispersion, so that in addition to the microchip, a drying
equipment is required for finally providing a dried material.
[0006] Analysis of the separated component is generally conducted
by mass spectrometry. For example, analysis using a MALDI-TOFMS
(Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass
Spectrometer) has been suggested as a method for efficiently
ionizing a polymer compound for mass spectrometry, and has been
applied to proteomics analysis (Patent document 2).
[0007] However, when a polymer compound analyzed by mass
spectrometry is a biological component such as a protein, a nucleic
acid or a polysaccharide, a target component must be isolated from
the biological sample in advance. For example, when analyzing a
sample comprising multiple components, the sample is purified and
then subjected to, for example, two-dimensional electrophoresis for
separating individual components; each component is collected from
each spot separated; and then the collected component is used to
prepare a sample for mass spectrometry. Thus, a separation and a
sample preparation processes must be separately conducted, leading
to a cumbersome procedure.
[0008] In a MALDI-TOFMS, a measurement sample is prepared by
blending a sample solution with a matrix solution and adding
dropwise the mixture to a metal-plate surface using an appropriate
tool such as a micropipette when using an ion-generation promoting
material called a matrix. Without a matrix, a sample solution is
applied dropwise to a plate in a similar manner.
[0009] FIG. 6 illustrates a conventional process for preparing a
sample for MALDI-TOFMS measurement. FIGS. 6(a) and 6(b) are a
cross-sectional view and a plan view, respectively, showing a
sample solution 131 dropped on the surface of a drying substrate
133. As shown in FIG. 6(b), the maximum width of the dropped sample
solution 131 is significantly larger than the maximum spot size 135
of a laser beam. As a result, a sample concentration per a unit
area is lower and thus, a relatively larger amount of sample is
required. The procedure is, therefore, not always a sample
preparation process suitable for analyzing a trace amount of sample
such as a biological component.
[0010] Furthermore, a sheet of drying substrate 133 is used for a
plurality of samples in a conventional method. Thus, a drying
process is needed for each sample. [0011] Patent Document 1:
Japanese Laid-Open Patent Publication No. 2002-207031 [0012] Patent
Document 2: Japanese Laid-open Patent Publication No.
1998-90226
SUMMARY OF THE INVENTION
[0013] As described above, a drying device has been needed, which
can efficiently concentrate and dry a small amount of sample such
as a biological sample. In particular, there has been needed a
drying device which can efficiently dry a collected sample for mass
spectrometry.
[0014] In view of the above situation, an objective of this
invention is to provide a small sample drying device capable of
conveniently and efficiently concentrating and drying a sample,
particularly a sample drying device capable of continuously and
efficiently drying a component prepared by processing, for example,
separation and purification, a biological sample.
[0015] Another objective of this invention is to provide a sample
drying device for mass spectrometry for efficiently concentrating
and drying a sample. A further objective of this invention is to
provide a mass spectrometer equipped with a drying device, which is
used as a substrate for sample drying and mass spectrometry.
[0016] According to this invention, there is provided a sample
drying device comprising a channel for a sample flowing in the
channel and a sample drying area having an opening communicating
with the channel, wherein the sample drying area comprises a fine
channel narrower than the channel.
[0017] In the sample drying device according to this invention, the
sample drying area has a narrower channel and an opening, so that a
sample in the channel is quickly guided to the sample drying area
by capillary phenomenon. The sample introduced in the sample drying
area is quickly dried. As the sample in the sample drying area is
dried, a sample solution in the channel is spontaneously and
continuously fed to the sample drying area. Thus, the drying device
of this invention can be easily operated and can efficiently dry
the sample.
[0018] In this invention, "fine channel(s)" may be formed as, for
example,
[0019] (i) voids between multiple protrusions formed in the drying
area or between filling members such as beads;
[0020] (ii) pores in a porous material disposed in the drying area;
or
[0021] (iii) concaves formed in the channel wall.
The fine channel preferably communicates with an opening. Thus, a
sample drying channel from the channel through the fine channel to
the opening can be ensured, so that the sample can be stably
dried.
[0022] According to this invention, there is also provided a sample
drying device comprising a main channel for a sample flowing in the
main channel; a plurality of side channels branched from the main
channel and a sample drying area communicating with the side
channels, wherein the sample drying area has a fine channel
narrower than the side channels.
[0023] In the sample drying device, the sample drying area is
formed in the side chain branched from the main channel, so that
the sample can be quickly dried. The side channel can be made
narrower than the main channel to ensure guiding a liquid from the
main channel to the side channel.
[0024] In the device having such a configuration, a sample can be
separated, prepared and/or analyzed as appropriate in the main
channel, then introduced into the side channel and finally dried in
the sample drying area. For example, the sample contains multiple
components and the main channel may comprise a separating portion
to separate the components. Such a configuration may allow the
individual components in the sample to be introduced to a plurality
of side channels for preparing dried materials of these components.
Thus, a single sample drying device can readily perform multiple
processes, for which multiple devices have been employed.
[0025] The sample drying device of this invention may comprise a
temperature controller for controlling a temperature of the sample
drying area. Thus, the sample drying area may be selectively heated
to continuously and more efficiently dry the sample and introduce
the sample from the channel to the sample drying area during the
sample drying.
[0026] In the sample drying device of this invention, the sample
drying area may comprise a plurality of protrusions separated each
other. A void between the protrusions becomes a fine channel, which
can ensure introduction of a liquid by capillarity to promote
sample drying.
[0027] The sample drying device of this invention may have a
configuration where the sample drying area may be filled with
multiple particles. Such a configuration may be easily formed by
filling the channel with the particles from an opening. Thus, a
narrower channel may be conveniently formed in the sample drying
area.
[0028] Alternatively, the sample drying device of this invention
may have a configuration where the sample drying area is filled
with a porous material. As used herein, the term "porous material"
refers to a structure having a fine channel communicating with the
outside in both sides.
[0029] The sample drying device of this invention may have a
configuration where the top of the sample drying area projects from
the opening. Thus, a surface area of the side wall of the sample
drying area may be further increased to further promote drying.
[0030] The sample drying device of this invention may have a
configuration where the sample drying area has a lid comprising a
fine channel communicating with the outside of the sample drying
device. The fine channel in the lid communicating with the outer
atmosphere allows a liquid to be guided from the channel to the
fine channel in the lid by capillary phenomenon, resulting in
efficient drying. Furthermore, since a dried sample is deposited
over the fine channel, a surface area of the dried sample can be
controlled by adjusting a width of the fine channel in the lid.
[0031] The sample drying device of this invention may have a
configuration where a metal film is formed on the surface of the
drying area. Thus, it may be suitable as an electrode for applying
an external force to an ionized sample when being used as a sample
holder in a mass spectrometer.
[0032] According to this invention, there is also provided a mass
spectrometer comprising a sample drying area included in the sample
drying device as a sample holder. Since the mass spectrometer of
this invention comprises the sample drying area as the sample
holder, the sample holder may be used as the sample drying device.
Thus, a pretreatment before conducting mass spectrometry, that is,
the steps of separation, purification, analysis and collection by
drying of components in a sample to be measure, may be continuously
conducted in the sample holder, resulting in improved operability.
A surface area of the dried sample may be adjusting by the size of
the opening over the sample drying area. Thus, the sample may be
formed into a shape corresponding to a spot system of a laser beam
applied to the sample during mass spectrometry. It can increase a
sample concentration in a laser irradiation area, to improve
accuracy and sensitivity of the measurement. Even in a small amount
of sample, a measurement sample can be, therefore, efficiently
prepared and analyzed.
[0033] According to this invention, there is also provided a mass
spectrometry system comprising separating unit separating
components in a biological sample by their molecular sizes and
properties; pretreatment unit pretreating the sample components
separated by the separating unit including enzymatic digestion;
drying unit drying the pretreated sample; and mass spectrometry
unit conducting mass spectrometry for the dried sample, wherein the
drying unit comprises the above sample drying device. Herein, the
biological sample may be obtained by extraction from an organism or
by synthesis.
[0034] As described above, this invention may provide a small
sample drying device for readily and efficiently concentrating or
drying a sample, which comprises a sample drying area having an
opening and a fine channel narrower than a channel. This invention
can also provide a sample drying device for mass spectrometry for
efficiently concentrating and drying a sample. This invention
further provides a mass spectrometer equipped with a drying device
used as a substrate for drying and mass spectrometry of a
sample.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] The above and other objects, features and advantages of the
present invention will be more apparent from the following
preferred embodiments and the accompanying drawings, in which:
[0036] FIG. 1 shows a configuration of a drying device according to
an embodiment of this invention;
[0037] FIG. 2 shows a configuration of a drying device according to
an embodiment of this invention;
[0038] FIG. 3 shows a configuration of a drying device according to
an embodiment of this invention;
[0039] FIG. 4 shows a configuration of a drying device according to
an embodiment of this invention;
[0040] FIG. 5 schematically shows a configuration of a microchip
according to an embodiment of this invention;
[0041] FIG. 6 illustrates a conventional method for preparing a
sample for mass spectrometry;
[0042] FIG. 7 is a process cross-sectional view illustrating a
process for manufacturing a drying device according to an
embodiment of this invention;
[0043] FIG. 8 is a process cross-sectional view illustrating a
process for manufacturing a drying device according to an
embodiment of this invention;
[0044] FIG. 9 is a process cross-sectional view illustrating a
process for manufacturing a drying device according to an
embodiment of this invention;
[0045] FIG. 10 illustrates a drying device according to an
embodiment of this invention when it is filled with a liquid;
[0046] FIG. 11 illustrates a change in a sample liquid when it is
heated by a heater in a drying device according to an embodiment of
this invention;
[0047] FIG. 12 schematically shows a configuration of a mass
spectrometer;
[0048] FIG. 13 is a block diagram of a mass spectrometry system
comprising a drying device according to an embodiment of this
invention;
[0049] FIG. 14 shows a configuration of a drying device according
to an embodiment of this invention;
[0050] FIG. 15 schematically shows a configuration of a chip
according to an embodiment of this invention;
[0051] FIG. 16 shows a configuration of a pillar disposed in a
drying area in a chip according to an embodiment of this
invention;
[0052] FIG. 17 illustrates DNA exudation in a drying area in a chip
according to Example; and
[0053] FIG. 18 illustrates a channel outlet in a drying area
without a pillar in a chip according to Example.
DETAILED DESCRIPTION OF THE INVENTION
[0054] This invention will be described by means of an exemplary
small drying device for readily and efficiently concentrating and
drying a sample. The drying device may be used as a sample holder
for a mass spectrometer such as a MALDI-TOFMS. In all of the
drawings, analogous components are designated by the same symbol,
whose description is omitted as appropriate.
First Embodiment
[0055] FIG. 1 shows a configuration of a drying device according to
this embodiment. FIGS. 1(a) and 1(b) are a plan view and a
cross-sectional view of a drying device 129, respectively.
[0056] In the drying device 129, substrate 101 comprises a channel
103, which comprises a drying area 107 having a plurality of
pillars 105 in one end. The channel 103 is covered by a cover 109,
but not covered by the cover 109, that is, opened in the drying
area 107. The bottom of the drying area 107 can be
temperature-controlled by a heater 111.
[0057] In the drying device 129, the drying area 107 comprises many
pillars 105. Thus, a sample liquid 141 can be charged such that it
wets the whole channel wall in the drying area 107. It will be
described with reference to FIG. 10. FIG. 10 illustrates a drying
device 129 filled with a liquid. FIG. 10(a) illustrates a drying
area 107 without pillars 105 while FIG. 10(b) illustrates a
configuration according to this embodiment.
[0058] As shown in FIG. 10(a), without a pillar 105, a sample
liquid 141 can wet only a part of the drying area 107 along a
channel wall from the cover 109. On the other hand, in FIG. 10(b),
there are provided pillars 105, whereby the sample liquid 141 is
introduced from a channel 103 to a drying area 107 by capillary
phenomenon and thus fills the whole drying area 107. Thus, in the
configuration in FIG. 10(b), the whole upper surface of the drying
area 107 can be covered by the sample liquid 141. Furthermore, the
pillars 105 ensure an adequate specific surface area in a channel
in the drying area 107. The drying device 129 having such a
configuration exhibits a higher drying efficiency.
[0059] The drying device 129 has a configuration where a sample
liquid introduced from the channel 103 to the drying area 107 by
capillary phenomenon is heated by a heater 111 to efficiently
evaporate a solvent. In the configuration shown in FIG. 10(b), the
pillars 105 on the channel 103 in the drying area 107 increases a
specific surface area of the channel in the sample drying area,
that is, a surface area of the wall per a volume of the sample
drying area, so that the sample can be quickly guided to the upper
surface and be efficiently concentrated in the drying area 107.
Then, the sample components are precipitated on the surface of the
drying area 107 and dried. Since the sample liquid 141 is
continuously fed from the channel 103 to the drying area 107,
operation is simple. In contrast, in the configuration shown in
FIG. 10(a), the sample liquid is in contact only with the bottom
and the sides of the channel 103, a heating efficiency is lower
than that in the configuration in FIG. 10(b).
[0060] A temperature of heating the drying area 107 by the heater
111 may be appropriately selected, depending on some factors such
as properties of components in the sample liquid to be dried; for
example, 50.degree. C. to 60.degree. C. both inclusive.
Alternatively, a drying rate of the sample liquid in the drying
area 107 may be 0.1 .mu.L/min to 10 .mu.L/min both inclusive, for
example, 1 .mu.L/min.
[0061] In the drying device 129, the lid 119 may have any shape by
which the substrate 101 can be covered such that at least part of
the upper part of the drying area 107 is opened. Since the channel
103 can be sealed by providing the cover 109, the sample liquid in
the channel 103 can be more efficiently guided into the drying area
107. Furthermore, the size of the opening can be adjusted to
control a shape of a dried sample as discussed in the sixth
embodiment later.
[0062] The substrate 101 is made of silicon. The silicon surface is
preferably oxidized. Thus, the substrate surface becomes
hydrophilic, so that a sample channel can be suitably formed.
Alternatively, the substrate 101 may be made of another material
such as a glass including quartz and a plastic. Examples of a
plastic include thermoplastic resins such as silicon resins, PMMA
(polymethylmethacrylate), PET (polyethyleneterephthalate) and PC
(polycarbonate) and thermosetting resins such as epoxy resins. Such
a material can be easily shaped, resulting in reduction in a
manufacturing cost for a drying device.
[0063] When using these materials, a metal film may be formed at
least over the whole surface of the drying area 107. A metal film
formed on the surface makes the device electro-conductive. Thus,
when a sample after drying is analyzed by mass spectrometry such as
MALDI-TOFMS as a whole drying device 129, a mass spectrometer may
be simplified because the drying area 107 can be used as an
electrode in the mass spectrometer for applying an electric
potential. Furthermore, it can prevent the component of the
substrate 101 from being sublimed along with a sample, to improve
accuracy and sensitivity in measurement.
[0064] The substrate 101 may be made of a metal. Using a metal, an
electric potential can be more stably applied by the drying area
107, when a sample after drying is analyzed by MALDI-TOFMS as a
whole drying device 129.
[0065] The pillars 105 may be, for example, formed by, but not
limited to, etching the substrate 101 in a predetermined
pattern.
[0066] The pillars 105 in FIG. 1 is cylindrical, but they may be,
in addition to a pillar or pseudo-pillar, a cone such as circular
cone and elliptic cone; a prism such as trigonal prism and
quadrangular prism; and pillars having another cross-sectional
shape. When the pillar 105 has a cross-sectional shape other than a
pseudo-circle, the pillar 105 may have an irregular side, resulting
in further increasing a surface area of the side and further
improving a liquid absorbing force by capillary phenomenon.
[0067] Alternatively, a slit having the cross-section in FIG. 1(a)
may be employed in place of the pillar 105. When using a slit, the
pillar 105 may have any of various shapes such as a striped
protrusion. Again, when using a slit, the side of the slit may be
irregular to further increase a surface area of the side.
[0068] In terms of the dimensions of the pillar 105, a width may
be, for example, about 5 nm to 100 .mu.m. In FIG. 1, a height is
substantially equal to the depth of the channel 103. Variation in a
height of the pillar 105 will be described in the forth
embodiment.
[0069] A distance between adjacent pillars 105 may be, for example,
5 nm to 10 .mu.m.
[0070] The cover 109 may be, for example, made of a material
selected from those for the substrate 101. The material may or may
not be the same as that for the substrate 101.
[0071] Next, there will be described a process for manufacturing a
drying device 129. The channel 103 or the pillars 105 may be formed
on the substrate 101 by, but not limited to, etching the substrate
101 into a predetermined pattern.
[0072] FIG. 7, FIG. 8 and FIG. 9 are process cross-sectional views
illustrating an exemplary manufacturing process. In sub-figures in
each figure, the middle is a top view and the right and the left
are cross-sectional views. In this process, the pillars 105 are
formed by the use of electron beam lithography using a calixarene
as a resist for fine processing. The following is an exemplary
molecular structure of a calixarene. A calixarene is used as a
resist for electron beam exposure and may be suitably used as a
resist for nano processing. ##STR1##
[0073] Herein, a substrate 101 is a silicon substrate with an
orientation of (100). First, as shown in FIG. 7(a), on the
substrate 101 are formed a silicon oxide film 185 and a calixarene
electron-beam negative resist 183 in sequence. Thicknesses of the
silicon oxide film 185 and the calixarene electron-beam negative
resist 183 are 40 nm and 55 nm, respectively. Then, an area to be
pillars 105 is exposed to an electron beam (EB). The product is
developed with xylene and rinsed with isopropyl alcohol. By this
step, the calixarene electron-beam negative resist 183 is patterned
as shown in FIG. 7(b).
[0074] Next, a positive photoresist 137 is applied to the whole
surface (FIG. 7(c)). Its thickness is 1.8 .mu.m. Then, the product
is developed by mask exposure such that the area to be the channels
103 is exposed (FIG. 8(a)).
[0075] Then, the silicon oxide film 185 is RIE-etched using a mixed
gas of CF.sub.4 and CHF.sub.3 to a thickness of 40 nm after etching
(FIG. 8(b)). After removing the resist by organic washing with a
solvent mixture of acetone, an alcohol and water, the substrate is
subjected to oxidation plasma treatment (FIG. 8(c)). Then, the
substrate 101 is ECR-etched using HBr gas. A height of the step in
the substrate 101 after etching, in other words, a height of the
pillars 105, is 400 nm (FIG. 9(a)). Next, the substrate is wet
etched with BHF-buffered hydrofluoric acid to remove the silicon
oxide film (FIG. 9(b)). Thus, the channel 103 and the pillars 105
are formed on the substrate 101.
[0076] Herein, it is preferable to make the surface of the
substrate 101 hydrophilic after the step in FIG. 9(b). By making
the surface of the substrate 101 hydrophilic, a sample liquid can
be smoothly guided into the channel 103 and the pillars 105. In
particular, in the drying area 107 where the channel is finer by
the pillars 105, hydrophilization of the channel surface is
preferable because it may promote introduction of a sample liquid
by capillary phenomenon to improve a drying efficiency.
[0077] After the step in FIG. 9(b), the substrate 101 is heated in
a furnace to form a silicon thermal oxide film 187 (FIG. 9(c)).
Herein, heating conditions are selected such that a thickness of
the oxide film becomes 30 nm. Forming the silicon thermal oxide
film 187 can eliminate difficulty in introducing a liquid into a
separating device. Then, a cover 189 is electrostatically joined.
After sealing, the drying device 129 is formed (FIG. 9(d)).
[0078] A metal film may be formed on the surface of the substrate
101. The metal film may be made of a material such as Ag, Au, Pt,
Al and Ti. It may be deposited by, for example, vapor deposition or
plating such as electroless plating.
[0079] When using a plastic material for the substrate 101, a known
method suitable for the type of the material for the substrate 101
may be employed, including etching, press molding using a mold such
as emboss molding, injection molding and photo-curing.
[0080] Again, when using a plastic material for the substrate 101,
the surface of the substrate 101 is preferably hydrophilized. By
hydrophilizing the surface of the substrate 101, a sample liquid
can be smoothly introduced into the channel 103 and the pillars
105. In particular, in the drying area 107 where the channel 103 is
finer by the pillars 105, hydrophilization of the surface of the
channel 103 is preferable because it may promote introduction of a
sample liquid 141 by capillary phenomenon to improve a drying
efficiency.
[0081] Surface treatment for hydrophilization may be, for example,
conducted by applying a coupling agent having a hydrophilic group
to the side wall of the channel 103. A coupling agent having a
hydrophilic group may be, for example, a silane coupling agent
having an amino group, more specifically;
N-.beta.(aminoethyl).gamma.-aminopropylmethyldimethoxysilane,
N-.beta.(aminoethyl).gamma.-aminopropyltrimethoxysilane,
N-.beta.(aminoethyl).gamma.-aminopropyltriethoxysilane,
.gamma.-aminopropyltrimethoxysilane,
.gamma.-aminopropyltriethoxysilane and
N-phenyl-.gamma.-aminopropyltrimethoxysilane. These coupling agents
may be applied by an appropriate method such as spin coating,
spraying, dipping and vapor deposition.
[0082] Again, in terms of FIG. 1, a heater 111 for controlling a
temperature of the drying area 107 is provided on the bottom of the
substrate 101 as shown in FIG. 1(b). By disposing the heater 111
such that the end of the drying area 107 is selectively heated, a
sample liquid can be surely introduced from the channel 103 to the
drying area 107 so that a drying efficiency in the drying area 107
can be further improved.
[0083] Heating of the drying area 107 is more preferably conducted
in an intermittent manner. FIG. 11 illustrates a change in a sample
liquid 141 during heating the drying area 107 by the heater 111. As
shown in FIG. 11(a), the drying area 107 is filled with the sample
liquid 141 and then heated by the heater 111. Then, drying proceeds
and the amount of the sample liquid in the drying area 107 is
reduced as shown in FIG. 11(b). When the heater is stopped after
drying proceeds to some extent, the drying area 107 is refilled
with the sample liquid (FIG. 11(a)). Then, the heater 111 is again
operated to restart drying (FIG. 11(b)). The procedure may be
repeated to conduct both drying and introduction of the sample
liquid in a balanced manner, resulting in improvement in a drying
efficiency.
Second Embodiment
[0084] FIG. 2(b) shows a configuration of a drying device according
to this embodiment. The configuration in FIG. 2(b) is as described
for the drying device of the first embodiment, except that a water
absorber 115 is formed in the drying area 107. The water absorber
115 has a surface having a relatively hydrophilic porous structure,
and a sample solution is introduced from the channel 103 to the
water absorber 115 filling the drying area 107 by capillary
phenomenon.
[0085] The water absorber 115 may have any shape where a sample
liquid can be introduced from the channel 103 to the drying area
107 by capillary phenomenon and evaporated on the surface. The
water absorber 115 may be, for example, porous silicon or porous
alumina with an etched concave structure formed by lithography.
Third Embodiment
[0086] FIG. 2(c) shows a configuration of a drying device according
to this embodiment. The configuration in FIG. 2(c) is as described
for the drying device in the first embodiment, except that the
drying area 107 is filled with beads 117. The beads 117 are fine
particles whose surface is relatively hydrophilic. A sample
solution is introduced from the channel 103 to the beads 117
filling the drying area 107 by capillary phenomenon.
[0087] The configuration in FIG. 2(c) can be provided by forming
the channel 103 in the surface of the substrate 101 as described in
the first embodiment and then filling one end of the surface with
the beads 117. Herein, since the upper part of the channel 103 is
opened, the configuration can be easily provided, because, for
example, the beads 117 can be smoothly placed.
[0088] The beads 117 may be made of any material whose surface is
relatively hydrophilic. In case of a highly hydrophobic material,
its surface may be hydrophilized. Examples of the material include
inorganic materials such as glasses and various organic and
inorganic polymers. The beads 117 may have any shape which, when
being placed, allows a channel for water to be ensured; for
example, particles, needles or plates. For example, the beads 117
as spherical particles may have an average particle size of 10 nm
to 20 .mu.m both inclusive.
[0089] Alternatively, the drying area 107 may be filled with metal
beads or semiconductor beads. Thus, an electric potential can be
more surely applied by the drying area 107, when a whole drying
device 129 is analyzed by mass spectrometry such as
MALDI-TOFMS.
[0090] Next, there will be described a method for filling the beads
117 in the channel 103. Before joining the cover 109, a mixture of
the beads 117, a binder and water is fed into the channel 103.
Herein, a damming member (not shown) is formed in the channel 103
to prevent the mixture from flowing outside the area to be the
drying area 107. Then, the mixture can be evaporated to dryness to
form the drying area 107.
[0091] A binder may be, for example, a sol containing a
water-absorbing polymer such as agarose gel and polyacrylamide gel.
A sol containing such a water-absorbing polymer can be used to
eliminate the need of drying because of spontaneous gelation.
Alternatively, the drying area 107 may be formed by filling the
channel 103 with a suspension of the beads 117 in water without a
binder and drying it under the atmosphere of dry nitrogen gas or
dry argon gas.
Fourth Embodiment
[0092] FIG. 3(c) shows a configuration of a drying device according
to this embodiment. The configuration of the drying device in FIG.
3(c) is as described in the first embodiment, except that the
pillars 105 protrude from the opening.
[0093] FIG. 3(a) shows a configuration where a height of the
pillars 105 is smaller than the depth of the channel 103; and FIG.
3(b) shows a configuration where a height of the pillars 105 is
substantially equal to the depth of the channel 103 as described in
the first embodiment. Since a surface area of the pillars 105
increases in the order of FIG. 3(a), FIG. 3(b) and FIG. 3(c), a
drying efficiency in the drying area 107 is improved. In the
configuration in FIG. 3(c), a sample is guided to the part above
the upper surface of the channel 103 by capillary phenomenon and
therefore a dried sample is also deposited in the upper part of the
channel 103. Thus, a dried target component can be more easily
collected. Since a sample is concentrated in a direction of the
height of the drying area 107, measurement can be more accurately
conducted in mass spectrometry such as MALDI-TOFMS.
Fifth Embodiment
[0094] FIG. 2(a) shows a configuration of a drying device according
to this embodiment. The configuration in FIG. 2(a) is as described
in the first embodiment, except that holes 113 are formed in the
drying area 107. While a target component is concentrated, dried
and deposited above the bottom of the channel 103 in the first to
the forth embodiments, the configuration in FIG. 2(a) is different
in that a target component is concentrated, dried and deposited at
the height near the bottom of the channel 103. In the configuration
where the holes 113 are formed in the drying area 107, a surface
area of the channel in the drying area 107 is also increased by the
holes 113, allowing a sample liquid to be efficiently concentrated
and dried.
[0095] The configuration in FIG. 2(a) can be provided as described
in the first embodiment, for example, by etching.
[0096] Although the holes 113 have a circular cross section in FIG.
2(a), it may have another shape such as a polygon. Furthermore, the
side of the hole 113 may be made convexoconcave to further increase
a surface area of the side of the hole 113 as described in the
first embodiment and to further increase a liquid absorbing force
by capillary decreasing.
[0097] The holes 113 may be a slit having the cross section in FIG.
2(a). When using a slit, a surface area of the side maybe also
further increased by making the slit side irregular.
[0098] The hole 113 may have, for example, a width of 10 nm to 20
.mu.m both inclusive and a depth of 10 nm to 20 .mu.m both
inclusive.
Sixth Embodiment
[0099] This embodiment relates to a drying device where a sample is
dried using an opening formed in the upper part of a channel as a
fine channel to deposit a dried sample on the upper surface of a
lid. FIG. 14 shows a configuration of a drying device according to
this embodiment. FIG. 14(a) is a top view of a drying device 143
and FIG. 14(b) is a cross-sectional view of the periphery of the
drying area 107 in FIG. 14(a). The drying device 143 comprises a
lid 119 covering the whole surface of the channel 103 including the
drying area 107. In the lid 119, an opening 121 is formed as a fine
channel, through which the channel 103 is communicated with the
outside air. Thus, a liquid in the sample introduced from the
channel 103 to the drying area 107 is guided to the opening 121 by
capillary phenomenon and then evaporated.
[0100] The lid 119 formed allows a dried sample 123 to be
selectively deposited near the opening 121 in the upper surface of
the lid 119. Furthermore, the size of the opening 121 can be
adjusted to adjust a surface area of the dried sample 123. One
opening 121 may be formed in the lid 119 as shown in FIG. 14, or
alternatively a plurality of openings 121 may be formed.
[0101] When forming the opening 121 in the lid 119 and, for
example, the drying device 143 and the dried sample 123 are
analyzed by MALDI-TOFMS measurement, the size of the dried sample
123 may be adjusted to be substantially equal to the maximum spot
size 135 of a laser beam described above in FIG. 6. Thus, a
concentration of the dried sample 123 can be increased in the
laser-beam irradiation site to improve accuracy and sensitivity in
measurement.
[0102] In the drying device 143, the pillars 105 may be formed in
the drying area 107 as described in the first embodiment, which is
shown in FIG. 4(a). Thus, the channel becomes finer in the drying
area 107, so that drying can be more efficiently conducted and the
dried sample 123 can be deposited near the opening 121 in the upper
surface of the lid 119 (FIG. 4(b)).
Seventh Embodiment
[0103] This embodiment relates to a microchip comprising a
plurality of the drying devices 127 described in the first
embodiment. FIG. 5 schematically shows a configuration of the
microchip according to this embodiment.
[0104] The microchip in FIG. 5 comprises a main channel 125 and a
plurality of side channels 127 branched from the main channel 125
on a substrate (not shown). Each side channel 127 is communicated
with a plurality of drying devices 129.
[0105] Using microchip in FIG. 5, a sample liquid containing
multiple components can be purified and separated into the
components, which can be finally concentrated, dried and collected
in the drying device 129.
[0106] For example, when a current is applied to the main channel
125 and the side channels 127 are filled with a gel and the like to
conduct separation similar to two-dimensional electrophoresis in
the microchip, the system can be designed such that a drying device
129 can be communicated with a site corresponding to a band for
each component separated in the side channel 127, to independently
collect each component from the sample.
[0107] Specifically, for separating water-soluble proteins in
blood, a separating device may be placed upstream of the main
channel 125 to remove insoluble components. Furthermore, a
separation mechanism which can remove low molecular weight
components in a plasma by permeation is employed to allow only high
molecular weight fractions to remain in the main channel 125. The
remaining high molecular weight fractions are two-dimensionally
separated in the main channel 125 and the side channels 127 as
described above, before introducing them into the drying device
129. Herein, the drying device 129 can be placed in the main
channel 125 upstream of the side channels 127 to concentrate the
high molecular weight fractions to some degree before separation
and thus to further improve a separation efficiency.
[0108] Although the drying device 129 is used in FIG. 5, a drying
device having another configuration according to this embodiment
may be, of course, employed.
Eighth Embodiment
[0109] In this embodiment, the drying device 129 according to the
first embodiment is used as a substrate for MALDI-TOFMS. There will
be described, as an example, preparation and measurement of a
protein sample for MALDI-TOFMS using the drying device 129.
[0110] For obtaining detailed data of a protein to be measured by
MALDI-TOFMS, its molecular weight must be reduced to about 1000 Da.
Thus, after molecular weight reduction, the sample is mixed with a
matrix solution and dried in the drying device 129 to provide a
dried sample.
[0111] When the target protein has an intramolecular disulfide
bond, the sample is subjected to reduction in a solvent such as
acetonitrile containing a reducing agent such as DTT
(dithiothreitol). Thus, a next decomposition reaction can
efficiently proceed. It is preferable that after reduction, a thiol
group is protected by, for example, alkylation to prevent
re-oxidation.
[0112] Next, the reduced protein molecule is subjected to molecular
weight reduction using a protein hydrolase such as trypsin. Since
molecular weight reduction is conducted in a buffer such as a
phosphate buffer, desalting and removal of the high molecular
weight fraction, that is, trypsin, must be conducted after the
reaction. The material obtained is mixed with a MALDI-TOFMS matrix
and introduced from the channel 103 to the drying area 107.
[0113] A temperature in the drying area 107 is controlled by the
heater 111 for concentrating and drying the sample to precipitate a
mixture of the matrix and the decomposed protein in the upper part
of the pillars 105. Herein, as described above in the first
embodiment, on-off of the heater 111 can be repeated for repeating
drying and introduction of the sample solution to efficiently
conduct drying.
[0114] After drying, the sample as a whole drying device 129 is set
in a MALDI-TOFMS apparatus. Then, while applying a voltage using
the drying device 129 as an electrode, for example, it is
irradiated with a nitrogen laser beam at 337 nm for MALDI-TOFMS
analysis.
[0115] There will be briefly described a mass spectrometer used in
this embodiment. FIG. 12 schematically illustrates a configuration
of the mass spectrometer. In FIG. 12, the dried sample is set on a
sample stage. Then, the dried sample is irradiated with a nitrogen
gas laser at a wavelength of 337 nm in vacuo, to vaporize the dried
sample together with the matrix. By applying a voltage using the
sample stage as an electrode, the vaporized sample travels in the
vacuum atmosphere and detected by a detection unit comprising a
reflector detector, a reflector and a linear detector.
[0116] Therefore, after fully drying the liquid in the drying
device 129, the drying device 129 can be placed in a vacuum chamber
in the MALDI-TOFMS apparatus and used as a sample stage for
MALDI-TOFMS. Since a metal film is formed on the surface of the
drying area 107 and is connectable to an external power source, a
potential can be applied to it as a sample stage.
[0117] Thus, using the drying device 129, the dried sample as the
whole drying device 129 can be used in MALDI-TOFMS. Furthermore,
for example, a sample separating device may be formed upstream of
the channel 103 to be able to conduct extraction, drying and
structural analysis of a target component on a single drying device
129. Such a drying device 129 may be useful in, for example,
proteome analysis.
[0118] Herein, since the drying device 129 is used as a chip for
MALDI-TOFMS, a step of washing an electrode plate for each sample
can be eliminated, resulting in improvement in operational
convenience and in measurement accuracy.
[0119] A MALDI-TOFMS matrix may be appropriately selected,
depending on a material to be measured. Examples of a matrix which
can be used include sinapic acid, .alpha.-CHCA
(.alpha.-cyano-4-hydroxycinnamic acid), 2,5-DHB
(2,5-dihydroxybenzoic acid), a mixture of 2,5-DHB and DHBs
(5-methoxysalicylic acid), HABA (2-(4-hydroxyphenylazo)benzoic
acid), 3-HPA (3-hydroxypicolinic acid), dithranol, THAP
(2,4,6-trihydroxyacetophenone), IAA (trans-3-indoleacrylic acid),
picolinic acid and nicotinic acid.
[0120] This embodiment has been described in terms of the drying
device 129 described in the first embodiment, but drying devices in
other embodiments can be, of course, used.
[0121] Alternatively, a fine-structure in the upper surface of the
drying area 107 comprising the pillars 105, the holes 113, the
water absorber 115 or the beads 117 and so forth in any of the
drying devices described in the above embodiments may be adjusted
to allow a sample to be more efficiently ionized without a matrix.
Such a configuration can eliminate the need for mixing a protein
solution with a matrix solution, so that, for example, each
fraction collected in the seventh embodiment together with the
drying device 129 may be used for MALDI-TOFMS.
[0122] FIG. 13 is a block diagram of a mass spectrometry system
comprising a drying device according to this embodiment. As shown
in FIG. 13(a), the system comprises means to perform each step of;
purification 1002 for removing impurities in a sample 1001 to some
degree; separation means 1003 for removing unnecessary components
1004; pretreatment 1005 for a separated sample; drying 1006 for a
sample after pretreatment; and identification 1007 by mass
spectrometry.
[0123] Drying by the drying device in this embodiment corresponds
to the drying step 1006, which is conducted on a microchip 1008.
The step of purification 1002 may be conducted, for example, using
a separating portion for separating only giant components such as
blood cells. The step of separation 1003 may be conducted by a
procedure such as two-dimensional electrophoresis, capillary
electrophoresis and affinity chromatography and so on. In the step
of pretreatment 1005, molecular weight reduction using, for
example, trypsin described above and mixing with a matrix are
conducted.
[0124] Since the drying device according to this embodiment
comprises a channel, the steps of purification 1002 to drying 1006
may be conducted on a piece of microchip 1008 as shown in FIG.
13(b). A sample may be continuously processed on the microchip 1008
to efficiently and reliably identify a trace amount of component in
a loss-reducing manner.
[0125] Thus, of the sample processing steps shown in FIG. 13, all
or those appropriately selected can be conducted on the microchip
1008.
[0126] This invention has been described with reference to some
embodiments. It will be understood by the skilled in the art that
these embodiments are only illustrative and that there may be many
variations for a combination of the components and the
manufacturing process, which are encompassed by the present
invention.
EXAMPLE
[0127] In this example, a drying device comprising the pillars
described above with reference to FIG. 1 was fabricated on a
substrate and evaluated. FIG. 15 schematically shows the drying
device. FIG. 15(a) is a top view of the drying device and FIG.
15(b) is a cross-sectional view taken on line A-A' of FIG.
15(a).
[0128] In FIG. 15, a channel 202 is formed on a substrate 201 and a
part of its upper surface is covered by a glass lid 203. The part
with the glass lid 203 is upstream while that without the lid is
downstream. A drying area 204 is formed in an outlet area in the
channel 202, in other words, the area upstream and downstream of
the end of the glass lid 203. The drying area 204 comprises
columnar structures 205.
[0129] In this example, the channel 202 and the columnar structure
205 were formed by the processing method described in the first
embodiment. Silicon was used as a substrate. The channel 202 had a
width of 80 .mu.m and a depth of 400 nm.
[0130] FIG. 16 shows a scanning electron microgram of the columnar
structure 205 formed in the outlet area in the channel 202. In FIG.
16 and FIGS. 17 and 18 described later, the lower direction from
the paper is upstream and the upper direction is downstream. As
shown in FIG. 16, the drying area 204 of the drying device of this
example comprises a plurality of strip-type columnar structures 205
with a width of 3 .mu.m aligned with an equal pitch of about 1
.mu.m in a longitudinal direction of the columnar structures 205 (a
transverse direction in this figure), and multiple rows of the
columnar structures 205 are disposed with an equal pitch of 700 nm
in a lateral direction of the columnar structures 205 (a vertical
direction in this figure). A height of the columnar structures 205
is 400 nm.
[0131] The drying device manufactured in this example was used to
continuously conduct drying and mass spectrometry of a DNA as
described below. The channel 202 was filled with a solution
containing a DNA (100 bp) stained with a fluorescent dye from the
upstream of the channel 202. Then, the outlet area in the channel
202 was observed by fluorescence microscopy. FIG. 17 shows a
fluorescence microgram of the area near the columnar structure 205
formed in the drying area 204 in the outlet area in the channel
202. FIG. 17 shows that the DNA brightly highlighted by the
fluorescence microscopy is exuded as a 60 .mu.m band downstream of
the glass lid 203. Thus, using the drying device of this example,
the sample could be stably introduced into the drying area 204 and
easily dried as described with reference to FIG. 10(b).
[0132] For comparison, a drying device without columnar structures
205 was manufactured in a similar manner. FIG. 18 shows a
fluorescence microgram for the device without columnar structures
205 in the outlet area in the channel, in which DNA is not exuded
outside of the glass lid 203. In the chip used in this example
without columnar structures 205 where the depth of the channel 202
is 400 nm, it can be seen that a wetting degree described with
reference to FIG. 10(a) is further reduced so that the drying area
204 is not wetted even in the area from the edge of the glass lid
203 to the wall surface of the channel 202.
[0133] Then, the DNA dried using the drying device in FIG. 17 was
analyzed by mass spectrometry. Specifically, the substrate 201 was
sonicated on an ultrasonic vibrator to fragmentate the DNA and then
the solvent was air dried. Then, a several microliters of matrix
was added dropwise to the dried DNA exuded in the outlet area in
the channel 202 and the product was analyzed by MALDI-TOFMS. As a
result, the analysis results from the DNA could be obtained.
[0134] As described above, in this example, the drying area 204
comprising a plurality of columnar structures 205 at the end of the
channel 202 whose upper surface is at least partly opened was
formed, so that the DNA could be moved to the drying area 204 and
then easily dried. Furthermore, the drying device could be used as
a sample stage for a mass spectrometer and mass spectrometry could
be conducted without removing the dried sample from the drying
device.
* * * * *